Project Summary/Abstract The lung possesses multiple stem/progenitor compartments along the proximal-distal axis where the epithelial stem/progenitors are situated in close proximity to the underlying stroma. Despite their homogeneity in appearance, it is increasingly apparent that the lung stroma contains diverse subsets, each uniquely suited to maintain the nearest stem/progenitor population. Thus, the current gap in knowledge is not whether stromal compartments are diverse, but rather how segregated stromal identities are maintained in different niches, and how disruption of distinct stromal identity can lead to disease. Utilizing a combination of single cell RNA-sequencing and a unique mouse genetic tool we built to isolate the stroma, our preliminary data demonstrate that hedgehog (Hh) activation promotes proximal stromal genes while suppressing genes associated with the distal alveolar stroma. Furthermore, distal expansion of Hh activation in the alveolar stroma attenuates stromal mitogen feedback to the alveolar stem/progenitors, leading to alveolar loss comparable to emphysema. Relevant to human disease, genome-wide association studies (GWASes) have identified numerous susceptibility loci for emphysema near the gene for Hedgehog-interacting protein (HHIP), a negative regulator of Hh activation, but mechanistic studies to define the pathogenic association have been lacking. Our central hypothesis is that HHIP, a negative regulator of SHH binding, restricts Hh activation to the proximal stroma to maintain proximal-distal segregation of stromal identity, the loss of which leads to disruption of the alveolar stem/progenitor niche and loss of alveoli comparable to emphysema. Leveraging the novel mouse genetic tools we have developed, our single cell analysis, and our access to clinical specimen, this proposal aims to address how stromal subsets maintain their distinct identity during normal homeostasis, how Hh alters the stromal feedback to the alveolar stem/progenitors, and how disruption of the alveolar niche can lead to chronic lung diseases such as emphysema.